Sporadic colorectal cancer (CRC) is one of the most frequently occurring and deadly of the oncological diseases affecting people in developed Western countries. It predominantly affects people over the age of 50.
A serious obstacle to early diagnosis of CRC is the absence of early, readily identifiable clinical manifestations in the majority of cases. It is only in the advanced stages of the disease, when larger tumours have formed, resulting in pain, bleeding and symptoms of obstruction, that the disease is readily diagnosed. However, the late stages of the disease are also associated with invasive or metastatic tumours. Thus, detection of colorectal tumours prior to the advanced stages of the disease would greatly increase the chances of successful surgical intervention and overall survival rates.
In the absence of early, readily identifiable clinical indications, the search for suitable CRC screening methods has continued for decades. Unfortunately, there is presently no CRC screening technique that combines low invasiveness, simplicity and low cost with high sensitivity and specificity. Two methods of screening for CRC are flexible colonoscopy/sigmoidoscopy and faecal occult blood testing (FOBT) [Rennert, G. Recent Results Cancer Res. 2003; 163: 248-253, Atkin, W. Scand. J. Gastroenterol. (Suppl.) 2003; 237: 13-16, Walsh, J. M. and Terdiman, J. P. JAMA 2003; 289: 1288-1296]. However, both of these methods have significant drawbacks.
Flexible colonoscopy/sigmoidoscopy is regarded as a precise and reliable diagnostic procedure, however, its invasiveness, cost and requirement for skilled and experienced specialists to carry out the procedure make its use in routine screening impractical. The same is true for recently introduced computed tomographic colonography (virtual colonoscopy).
FOBT is cheap and simple, however, it produces unacceptably high rates of both false negative and false positive results. Despite these limitations, FOBT is presently the screening method of choice.
Alternative methods of diagnosing CRC based upon a direct indicator of tumour presence have been investigated. One indicator that has been identified is analysis of exfoliated colonocytes. Exfoliation of colonocytes (i.e. spontaneous detachment of cells from orderly organized epithelial layer of colonic mucosa) is an important cell renewal mechanism in the human gut [Eastwood, G. L. Gastroenterology 1977; 41: 122-125]. Cytological analysis of colonocytes obtained from colonic or rectal washings (i.e. by irrigation of the colorectal mucosa) was carried out approximately 50 years ago [Bader, G. M. and Papanicolau, G. N. Cancer 1952; 5: 307-14]. This work showed that morphologically distinct exfoliated neoplastic cells could be detected in CRC patients. However, the method of obtaining these samples (an invasive colonic lavage procedure) suffered from the same disadvantages as sigmoidoscopy/colonoscopy, and required detailed cytological analysis of the sample once obtained.
The prevailing approach to obtaining samples of exfoliated epithelial cells has been to isolate them from human faeces. Human faeces were identified as a source of such cells, as the exfoliated cells of the colonic epithelium can be excreted in conjunction with other faecal matter.
The first attempts to use colonocytes isolated from human faeces for diagnostic and research purposes were started about 15 years ago by P. P. Nair and his colleagues. They claimed to be able to recover thousands of “viable” exfoliated cells from a few grams of dispersed faecal material using an isolation procedure based on density gradient centrifugation [Iyengar, V. et al., FASEB J 1991; 5: 2856-2859, Albaugh, G. P. et al., Int. J. Cancer 1992; 52: 347-350]. However, these ambitious claims have generated substantial doubts due to the low likelihood of the presence of well-preserved colonocytes in an aggressive anaerobic environment such as that found in the faeces. Furthermore, morphological evidence presented in their 1991 reference was unconvincing. P. P. Nair and members of his group maintain the validity of their approach [Nair, P. et al., J. Clin. Gastroenterol. 2003; 36 (5 Suppl.) S84-S93], but have not produced any practical advances based on the outcomes of their studies.
However, despite the lack of practical advances by P. P. Nair and his colleagues, the use of human stool for diagnostic and research purposes remains an active research area as it is not associated with any invasive intervention. A number of groups have undertaken attempts to isolate colonocyte-derived genetic material (DNA) from human stool samples in order to develop diagnostic procedures employing molecular biomarkers of malignancy. Whilst DNA directly isolated from homogenized faeces can be amplified and analysed for the presence of cancer-associated genetic alterations, the absence of a highly reliable single molecular biomarker for cancer resulted in the use of multiple molecular markers reflecting a number of genetic alterations known to be present in malignant cells at relatively high frequencies. Several approaches proposing simultaneous detection of multiple mutations in the APC, K-ras and p53 genes combined with microsatellite marker analysis have been described [Ahlquist, D. A. et al., Gastroenterology 2000; 119: 1219-1227, Dong, S. M. et al., J. Natl. Cancer Inst. 2001; 93: 858-865, Rengucci, C. et al., Clin. Cancer Res. 2001; 93: 858-865, Traverso, G. et al., N. Engl. J. Med. 2002; 346: 311-320]. Methylation changes in faecal DNA have also been considered as a potential diagnostic marker [Muller, H. M. et al., Lancet 2004; 363: 1283-1285]. Although detection of colorectal tumours by multi-target molecular assays appears to be feasible, the validity of these methods for screening purposes remains questionable due to the high cost and relative complexity of laboratory procedures involved.
The search for CRC molecular markers in DNA extracted from homogenized stool samples has overshadowed the importance of the initial collection/isolation of exfoliated colonocytes. It is, however, apparent that homogenized stool is a difficult material for human DNA extraction. In particular, the abundance of bacteria in faeces can interfere with colonocyte DNA recovery procedures, and rapid mammalian DNA damage and degradation occur in the presence of anaerobic bacterial flora of the human colon.
The development of approaches based upon exfoliated colonocyte isolation has been slow partially due to a surprising lack of knowledge on cell exfoliation in the gut both in normal physiological conditions and in disease. The current views on colonocyte exfoliation are still affected by an old and unproven hypothesis implying “obligatory” exfoliation of nearly all differentiated colonocytes upon their migration to the luminal epithelium from the colonic crypts (i.e. it is presumed that there should be millions of colonocytes present in faecal matter because the cell proliferation rate of colonic epithelium is high and all cells are eventually exfoliated). It is, however, becoming clear that programmed cell death or apoptosis in situ is at least as important as exfoliation [Hall, P. A. et al., J. Cell Sci. 1994; 107: 3569-3577, Barkla, D. H. and Gibson, P. R. Pathology 1999; 31: 230-238, Ahlquist, D. A. et al., Hum. Pathol. 2000; 31: 51-57]. The relationship between these two major mechanisms of cell removal from colonic mucosa may undergo significant changes in colorectal neoplasia [Ahlquist, D. A. et al. (supra)]. Indeed, it is now proven that normal regulatory pathways leading cells to apoptosis are severely deregulated in malignant tumours [Bedi, A. et al., Cancer Res. 1995; 55: 1811-1816, LaCasse, E. C. et al., Oncogene 1998; 17: 3247-3259, Jass, J. R. Gastroenterology 2002; 123: 862-876, Oren, M. Cell Death Differ. 2003; 10: 431-442, Boedefeld, W. M. 2nd et al., Ann. Surg. Oncol. 2003; 10: 839-851] resulting in a greatly reduced apoptotic potential of cancer cells. At the same time, tumour cell adhesion is known to diminish dramatically as cancer progresses [Yamamoto, H. et al., Cancer Res. 1996; 56: 3605-3609, Haier, J. and Nicolson, G. L. Dis. Colon Rectum 2001; 44: 876-884, Leeman, M. F. et al., J. Pathol. 2003; 201: 528-534]. The latter phenomenon is important for metastatic spread, however in colorectal neoplasia, combined suppression of apoptosis and decrease in intercellular adhesion/communication greatly increases the chances of malignant cell shedding from the surface of growing tumours. If this is the case, exfoliated tumour cells, some of which can probably retain proliferative potential, should differ from their normal (non-tumour) exfoliated counterparts in: i) being more abundant due to facilitated exfoliation from the tumour surface; and ii) having much greater “survival” capacity, in particular due to higher resistance to the lack of oxygen [Graeber, T. G. et al., Nature 1996; 379: 88-91]. Upon exfoliation, these cells enter a relatively well oxygenated “mucocellular layer” that separates the colonic mucosa from the faecal contents of the gut and permanently moves distally with the flow of faeces [Ahlquist, D. A. et al. (supra)].
The importance of the mucocellular layer providing an interface between colorectal mucosa and faecal contents of the gut has not been understood until recently. Experimental studies indicated that good quality DNA could, be easily obtained from the surface of rat faeces and used for further amplification and gene mutation analysis [Loktionov, A. and O'Neill, I. K. Int. J. Oncol. 1995; 6: 437-445]. These early experiments suggested that DNA extracted from colonocytes isolated from human stool surface (stool surface can be regarded as a fraction of mucocellular layer excreted with faeces) could be used for molecular analysis. A method of exfoliated cell isolation from human whole stool samples by washing cells off the surface of cooled faeces and collecting them by immunomagnetic separation procedure has been developed [Loktionov, A. et al., Clin. Cancer Res. 1998; 4: 337-342]. Although work in this direction was initially planned in terms of developing a molecular diagnostic assay for CRC, it emerged that a simple quantitative analysis of colonocyte-derived DNA from human stool surface could be used for CRC diagnosis and screening since the relative DNA amount in CRC patients was much higher compared to healthy individuals. Other authors have also reported higher amounts of either exfoliated cells [Dutta, S. K. et al., Gastroenterology 1995; 108 (Suppl.): A463] or DNA [Villa, E. et al., Gastroenterology 1996; 110: 1346-1353] in dispersed or homogenized stool samples obtained from CRC patients, however the differences between healthy people and cancer patients observed in those studies were not large enough to be considered diagnostically valid. By contrast, Loktionov et al (supra) were able to show the existence of a striking difference between CRC patients and healthy individuals employing a calculated index relating to the amount of DNA extracted from cells isolated from the stool surface to stool weight (stool DNA index or SDNAI).
The SDNAI-based diagnostic method is described in U.S. Pat. No. 6,187,546. Although the technique and results of its initial trials apparently highlighted a very efficient, simple and inexpensive approach to CRC screening, it had a number of substantial faults (apparent difficulties of whole stool handling and especially impossibility of the procedure standardization) preventing its commercialization and serious introduction into clinical practice. It has also become clear that relatively small numbers of well-preserved cells can be obtained from human stool surface using this technique [Bandaletova, et al., APMIS 2002; 110: 239-246]. These problems, difficulty of standardization being the crucial one, cause serious doubts with regard to using exfoliated colonocytes isolated from stool samples for wide scale CRC screening.
There is a good body of evidence indicating that the mucocellular layer covering human rectal mucosa is particularly rich in well-preserved exfoliated colonocytes. In addition, the cellular content of this layer in CRC patients appears to be much higher than in healthy individuals, primarily due to the greatly increased presence of highly resistant malignant colonocytes. Therefore, CRC patients' tumor cells, which are much better adapted to autonomous existence, should quantitatively dominate the rectal exfoliated cell pool. Several recent reports describing distal (e.g. anal) implantation of persisting exfoliated cells from removed colorectal tumors [Jenner, D. C. et al., Dis. Colon Rectum 1998; 41: 1432-1434, Wind, P. et al., Dis. Colon Rectum 1998; 41: 1432-1434, Isbister, W. H., Dig. Surg. 2000; 17: 81-83, Hyman, N. and Kida, M., Dis. Colon Rectum 2003; 46: 835-836, Abbasakoor, F. et al., Ann. R. Coll. Surg. Engl. 2004; 86: 38-39] strongly corroborate this hypothesis.
Direct access to the rectal mucosa is possible by routine digital rectal examination with an examiner's gloved finger. However, although one can achieve a contact with the rectal mucocellular layer by employing this simple manipulation, significant losses of material and simultaneous contamination with irrelevant squamous epithelium of the anal canal are inevitable during the removal of the finger from the rectum. Smears prepared from gloves used for rectal examination have shown well-preserved colonocytes, combined with a high level of contamination by cells of the squamous epithelium.
There is thus a great need for direct collection of exfoliated epithelial cells from the surface of rectal mucosa without the problems of material loss and serious contamination with other tissue elements at the stage of removal of the cell-collecting surface from the rectum. Such cells could be used not only for quantitative cell and DNA analysis, but also investigated for the presence of additional cancer biomarkers (e.g. proteins) and finally assessed immunohistochemically and cytologically.